CN106591309A - Theophylline induction type gene expression system - Google Patents

Theophylline induction type gene expression system Download PDF

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Publication number
CN106591309A
CN106591309A CN201611112241.0A CN201611112241A CN106591309A CN 106591309 A CN106591309 A CN 106591309A CN 201611112241 A CN201611112241 A CN 201611112241A CN 106591309 A CN106591309 A CN 106591309A
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theophylline
expression
gene expression
bacillus
riboe1
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周哲敏
崔文璟
程锦涛
韩来闯
周丽
刘中美
索菲娅
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/002—Vectors comprising a special translation-regulating system controllable or inducible

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Abstract

The invention discloses a theophylline induction-type gene expression system, which belongs to the technical field of microbial biomass. The system takes a composition-type strong promoter P43 as a basic component, merges a theophylline controlling element riboE1 with reconstructed interval zone length among an artificial SD element and a SD sequence and an initial code, constructs the theophylline induction-type gene expression element P43-riboE1, and clones a sequence to a bacillus subtilis expression vector pBSG, the induction expression carrier pBSG11 is constructed, host 168 bacterium is conversed, and high-level and high inductivity expression of exogenous gene under theophylline control is realized. The high-efficiency controllable recombination expression of report gene GFP, GUS and aspartase is realized by using the recombinant bacteria. Compared with inductive agents such as xylose and IPTG, the system has the advantages of no toxicity, and low cost, and has wide application space by taking theophylline as the gene expression system of the inductive agent.

Description

A kind of theophylline inducible gene expression system
Technical field
The present invention relates to a kind of theophylline inducible gene expression system, belongs to field of microbial biotechnology.
Background technology
Inducible gene expression has important application in protokaryon and eucaryon allogeneic gene expression system.At present in hay In bacillus cereuss commonly use inducible gene expression element have hydridization by isopropyl-beta D-thio galactopyranoside (IPTG) The Pgrac promoteres of induction and the PxylA promoteres of xylose induced activation.Although these gene expression elements can realize external source base Because of the expression in bacillus subtilises, but affected by Gene regulation mode, these promoteres are required for importing one simultaneously The repressor protein of external source, could realize the transformation of tranquillization-induction.Increased and built in bacillus subtilises using these systems The complexity of recombination system.And need to add in incubation using the Primary structure system of above-mentioned promoter element Plus respective required chemical agent ability activated gene transcription, start protein translation, therefore be difficult to realize preparing on a large scale.
The content of the invention
Present invention firstly provides it is a kind of control gene expression element, its nucleotide sequence as shown in SEQ ID NO.1, It is P43 promoteres fusion theophylline ribosome switch control element, and the strong SD of synthetic is have matched in ribosome switch downstream The gene order that sequence is obtained.
The present invention also provides a kind of expression vector, and its promoter is P43- of the nucleotide sequence as shown in SEQ ID NO.1 riboE1。
In one embodiment of the invention, the expression vector is E.coli-B.subtilis shuttle vector, Its promoter is P43-riboE1 of the nucleotide sequence as shown in SEQ ID NO.1.
The present invention also provides a kind of theophylline induction type allogeneic gene expression system, includes and is made up of by force bacillus subtilises Type Promoter P43 has merged theophylline ribosome switch in downstream, and have matched the strong SD of synthetic in ribosome switch downstream Sequence, the gene expression element P43-riboE1 of the new theophylline ribosome switch-mode regulation for obtaining.
The present invention provides a kind of artificial constructed novel protein expression control element-theophylline ribosome switch, this unitary After part transcription, the loop-stem structure formed in 5 ' untranslated regions closes ribosome binding element SD wherein, when with the presence of theophylline When, theophylline is combined in the inside of loop-stem structure, changes stem ring base pairing, SD is discharged, so as to complete turning over for recombinant protein Translate.This inducible expression system adjusts the expression of foreign protein using the special construction at the ends of mRNA 5 ' in translation skill, can Using various constitutive promoters, it is not necessary to additionally introduce the transcription of repressor protein regulator gene, greatly simplify in hay spore The difficulty of expression of recombinant proteins system is built in bacillus, and suitable for building the system for extensive Prepare restructuring albumen.
Description of the drawings
Fig. 1. the structure schematic diagram of the GFP gene expression systems containing theophylline riboswitch element.
Fig. 2 .0.5,1,2,4, GFP expressions, the detection of inductivity under the theophylline inductive condition of 8mM；(a) variable concentrations Theophylline induction pBSG11 express in bacillus subtilis B.subtilis168 GFP level change (sample treatment, applied sample amount are equal It is identical)；relative fluorescent density：Relative fluorescent intensity, represents that unit thalline expresses the level of GFP；(b) The theophylline induction pBSG11 of variable concentrations expresses the change of GFP inductivities；induction ratio：Inductivity, through induction Unit thalline expresses the level of GFP during the level/do not induce of unit thalline expression GFP；C () SDS-PAGE detects variable concentrations tea The change (sample treatment, applied sample amount all same) of the amount of alkali induction pBSG11 expression GFP.
Fig. 3 SDS-PAGE detect P43 promoter constitutive expression GFP and the expression by GFP after the induction of 8mM theophylline (sample treatment, applied sample amount all same).
After Fig. 4 .8mM theophylline induction 16h, 20h, GUS expressions and enzyme activity；A () SDS-PAGE detection 8mM theophylline is lured After leading recombination bacillus subtilis B.subtilis/p43E-gus expression gus protein 16h and 20h, the expression of gus protein is (at sample Reason, applied sample amount all same), it is expressed as 0mM through the matched group thalline of 4% dimethyl sulfoxide process；(b) recombinant bacterium The activity of B.subtilis/p43E-gus gus proteins after 8mM theophylline induces 16h and 20h；induction ratio：Induction Rate, GUS is active after the induction of 8mM theophylline/and do not induce matched group GUS active；GUS activity：GUS is active.
Specific embodiment
Embodiment 1
(1) B.subtilis/pBSG11 and B.subtilis/p43E-gus recombinant bacteriums are built
P43-riboE1 gene of the sequence after optimization as shown in SEQ ID NO.1 is carried out into full genome synthesis, Jing After BamHI/SalI enzyme action, in being cloned into pUC19 plasmids, pUC57A plasmids are obtained.With pUC57A as template, P43- is expanded RiboE1 sequences, as big primer after recovery, insert the srfA promoteres position of pBSG03 plasmids, replace srfA promoteres, Construct pBSG11 recombiant plasmid.PBSG11 recombinant plasmid transformeds B.subtilis 168, obtain recombinant bacterium B.subtilis/ pBSG11.The construction method of pBSG03 is referring to document Chengran Guan, Wenjing Cui*, Jiantao Cheng, Li Zhou,Junling Guo,Xu Hu,Guoping Xiao,Zhemin Zhou*.Construction and development of an auto-regulatory gene expression system in Bacillus subtilis.Microb Cell Fact.2015,14(1):150。
With primer gus F-homo：
CAAAACCCCCCTTTGCTGAGGTGGCAGAGGGCAGGTATGATAGGTGGTATGTTTTCGCTTGAACTTT； gus R-homo：
AGGCCGTCGAGTTTTTTGATTTCACGGGTTGGGGTTTCTACAGGACGTAACATCTTGTTGTTACCTCCTTTAGCAGG GTGCTG；Gus genetic fragments are amplified from E. coli JM109, PCR fragment is reclaimed, using pBSG11 as mould Plate, by the use of the gus genetic fragments for reclaiming as big primer, the position of GFP genes gus genes being inserted in pBSG11, together When replace GFP genes.
PCR system：The μ L of pBSG11 templates 0.5, the μ L of PCR fragment 1.5, Takara primerSTAR Max DNA The μ L of polymerase 12.5, ddH2O 10 μ L.PCR conditions：95 DEG C of 5min, 95 DEG C of 1min, 58 DEG C of 20s, 72 DEG C of 4min.Amplification Product is digested through 1 μ L DpnI, converts e. coli jm109, and picking positive colony extracts plasmid and surveyed after culture Sequence checking, obtains p43E-gus recombiant plasmid.After conversion B.subtilis 168, recombinant bacterium B.subtilis 168/ is obtained P43E-gus, is expressed as BSG-GUS.
(2) B.subtilis/pBSG11 recombinant bacteriums receive theophylline induced expression level
Incubated overnight recombinant bacterium B.subtilis/pBSG11 in LB, in being seeded to 250mL triangular flasks, cultivates to OD600About During equal to 3, be separately added into 0.5,1,2,4, the theophylline of 8mM.After induction 24h, collects thalline a, part is used to determine fluorescent value, A part is for SDS-PAGE detections GFP expressions after smudge cellses.
As shown in Fig. 2 the GFP expressions of recombinant bacterium B.subtilis/pBSG11 are continuous with the increase of theophylline concentration Improve, and when processing without theophylline derivant, the GFP expressions of recombinant bacterium B.subtilis/pBSG11 are very low, finally measure Both ratio --- inductivity is 8.6.As shown in figure 3, the P43 promoter constitutive expressions before SDS-PAGE detection transformations GFP and shown that the P43-riboE1 sequences that the present invention is provided are compared by the result of expression of GFP after the induction of 8mM theophylline P43 promoteres, can significantly improve the expression of target protein.
(3) with same method culture B.subtilis/p43E-gus recombinant bacterium BSG-GUS, as shown in figure 4, Jing 8mM After theophylline induction 24h, gus great expressions, enzyme activity level reaches 1.8U/mL.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention Enclosing should be by being defined that claims are defined.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A kind of theophylline inducible gene expression system
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 356
<212> DNA
<213>Artificial sequence
<400> 1
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60
taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120
tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240
gcgattatgt aaaatataaa gtgatagcgg taccattata atacgactca ctataggtga 300
taccagcatc gtcttgatgc ccttggcagc accctgctaa aggaggtaac aacaag 356
<210> 2
<211> 67
<212> DNA
<213>Artificial sequence
<400> 2
caaaaccccc ctttgctgag gtggcagagg gcaggtatga taggtggtat gttttcgctt 60
gaacttt 67
<210> 3
<211> 83
<212> DNA
<213>Artificial sequence
<400> 3
aggccgtcga gttttttgat ttcacgggtt ggggtttcta caggacgtaa catcttgttg 60
ttacctcctt tagcagggtg ctg 83

Claims (10)

1. it is a kind of control gene expression element, it is characterised in that its nucleotide sequence is as shown in SEQ ID NO.1.
2. a kind of expression vector, it is characterised in that its gene expression control elements is nucleotide sequence as shown in SEQ ID NO.1 P43-riboE1.
3. a kind of expression vector according to claim 2, it is characterised in that the expression vector is E. coli-B Bacillus cereuss shuttle vector, its original promoter replaces with the P43-riboE1 for nucleotide sequence as shown in SEQ ID NO.1.
4. a kind of Bacillus subtilis genes expression system, it is characterised in that its gene expression control elements is nucleotide sequence P43-riboE1 as shown in SEQ ID NO.1.
5. a kind of Bacillus subtilis genes expression system according to claim 4, it is characterised in that expressive host is withered Careless bacillus cereuss.
6. a kind of Bacillus subtilis genes expression system according to claim 4 or 5, it is characterised in that expression vector For E.coli-B.subtilis shuttle vector.
7. it is a kind of prepare described in claim 1 control gene expression element method, it is characterised in that be in hay spore The strong constitutive promoter P43 of bacillus matches strong SD sequences in downstream fusion theophylline ribosome switch in ribosome switch downstream, The gene expression element P43-riboE1 of the new theophylline ribosome switch-mode regulation for obtaining.
8. a kind of method of application bacillus subtilises expressing protein, it is characterised in that using the control base described in claim 1 Because of the element expressed.
9. a kind of method of application bacillus subtilises expressing protein, it is characterised in that carry using the expression described in claim 2 Body.
10. a kind of method of application bacillus subtilises expressing protein, it is characterised in that arbitrary described using claim 4～6 Bacillus subtilis genes expression system.
CN201611112241.0A 2016-12-07 2016-12-07 Theophylline induction type gene expression system Pending CN106591309A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109811010A (en) * 2019-01-15 2019-05-28 浙江大学 A kind of method and its application enhancing actinomyces gene editing efficiency
CN109852650A (en) * 2018-12-18 2019-06-07 江南大学 A kind of artificial aptamer enzyme and application by theophylline regulation
CN110592080A (en) * 2018-12-17 2019-12-20 中国科学院天津工业生物技术研究所 Optimized maltose promoter mutant and application thereof
CN111269930A (en) * 2020-02-14 2020-06-12 江南大学 Method for detecting genetic stability of filamentous fungus transformation system
CN114107309A (en) * 2021-11-19 2022-03-01 江南大学 Non-natural theophylline RNA molecular switch

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CUI，W.J.等: "Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch", 《 MICROBIAL CELL FACTORIES》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110592080A (en) * 2018-12-17 2019-12-20 中国科学院天津工业生物技术研究所 Optimized maltose promoter mutant and application thereof
CN110592080B (en) * 2018-12-17 2021-11-23 中国科学院天津工业生物技术研究所 Optimized maltose promoter mutant and application thereof
CN109852650A (en) * 2018-12-18 2019-06-07 江南大学 A kind of artificial aptamer enzyme and application by theophylline regulation
CN109811010A (en) * 2019-01-15 2019-05-28 浙江大学 A kind of method and its application enhancing actinomyces gene editing efficiency
CN111269930A (en) * 2020-02-14 2020-06-12 江南大学 Method for detecting genetic stability of filamentous fungus transformation system
CN114107309A (en) * 2021-11-19 2022-03-01 江南大学 Non-natural theophylline RNA molecular switch
CN114107309B (en) * 2021-11-19 2023-07-25 江南大学 Non-natural theophylline RNA molecular switch

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